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Functional characterization of human cancer-derived TRKB mutations.

Geiger TR, Song JY, Rosado A, Peeper DS - PLoS ONE (2011)

Bottom Line: Unexpectedly, both colon cancer-derived mutants, TRKB(T695I) and TRKB(D751N), displayed reduced activity compared to that of wild-type TRKB.Consistently, upon stimulation with the TRKB ligand BDNF, these mutants were impaired in activating TRKB and its downstream effectors AKT and ERK.In conclusion, we fail to detect any gain-of-function of four cancer-derived TRKB point mutations.

View Article: PubMed Central - PubMed

Affiliation: Division of Molecular Genetics, Netherlands Cancer Institute, Amsterdam, the Netherlands.

ABSTRACT
Cancer originates from cells that have acquired mutations in genes critical for controlling cell proliferation, survival and differentiation. Often, tumors continue to depend on these so-called driver mutations, providing the rationale for targeted anticancer therapies. To date, large-scale sequencing analyses have revealed hundreds of mutations in human tumors. However, without their functional validation it remains unclear which mutations correspond to driver, or rather bystander, mutations and, therefore, whether the mutated gene represents a target for therapeutic intervention. In human colorectal tumors, the neurotrophic receptor TRKB has been found mutated on two different sites in its kinase domain (TRKB(T695I) and TRKB(D751N)). Another site, in the extracellular part of TRKB, is mutated in a human lung adenocarcinoma cell line (TRKB(L138F)). Lastly, our own analysis has identified one additional TRKB point mutation proximal to the kinase domain (TRKB(P507L)) in a human melanoma cell line. The functional consequences of all these point mutations, however, have so far remained elusive. Previously, we have shown that TRKB is a potent suppressor of anoikis and that TRKB-expressing cells form highly invasive and metastatic tumors in nude mice. To assess the functional consequences of these four TRKB mutations, we determined their potential to suppress anoikis and to form tumors in nude mice. Unexpectedly, both colon cancer-derived mutants, TRKB(T695I) and TRKB(D751N), displayed reduced activity compared to that of wild-type TRKB. Consistently, upon stimulation with the TRKB ligand BDNF, these mutants were impaired in activating TRKB and its downstream effectors AKT and ERK. The two mutants derived from human tumor cell lines (TRKB(L138F) and TRKB(P507L)) were functionally indistinguishable from wild-type TRKB in both in-vitro and in-vivo assays. In conclusion, we fail to detect any gain-of-function of four cancer-derived TRKB point mutations.

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Responsiveness of human cancer-derived TRKB mutants to BDNF.(A) RIE-1 cells expressing wild-type or mutant TRKB were stimulated with 10 ng/ml recombinant BDNF and analyzed for phospho-tyrosine (pY) and total TRK content by immunoprecipitation (IP) and subsequent immunoblot (IB) analysis. (B) RIE-1 cells expressing wild-type or mutant TRKB were stimulated with 1 ng/ml recombinant BDNF and analyzed for phospho (p) and total TRK. (C) RIE-1 cells expressing wild-type or mutant TRKB were stimulated with 10 ng/ml recombinant BDNF and analyzed for phospho- (p) AKT and total AKT. PI3K inhibitor LY294002 was applied to confirm the identity of the pAKT signal indicated by the arrowhead. Asterisk (*) indicates a non-specific band. The samples loaded in lanes two and three serve as controls and were derived from a replicate experiment performed under identical conditions. (D) RIE-1 cells expressing wild-type or mutant TRKB were stimulated with 1 ng/ml recombinant BDNF and analyzed for phospho- (p) ERK and total ERK. For all panels, the numbers underneath indicate quantification of the phospho-specific signals, normalized to the total signals and relative to those of wild-type TRKB.
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pone-0016871-g004: Responsiveness of human cancer-derived TRKB mutants to BDNF.(A) RIE-1 cells expressing wild-type or mutant TRKB were stimulated with 10 ng/ml recombinant BDNF and analyzed for phospho-tyrosine (pY) and total TRK content by immunoprecipitation (IP) and subsequent immunoblot (IB) analysis. (B) RIE-1 cells expressing wild-type or mutant TRKB were stimulated with 1 ng/ml recombinant BDNF and analyzed for phospho (p) and total TRK. (C) RIE-1 cells expressing wild-type or mutant TRKB were stimulated with 10 ng/ml recombinant BDNF and analyzed for phospho- (p) AKT and total AKT. PI3K inhibitor LY294002 was applied to confirm the identity of the pAKT signal indicated by the arrowhead. Asterisk (*) indicates a non-specific band. The samples loaded in lanes two and three serve as controls and were derived from a replicate experiment performed under identical conditions. (D) RIE-1 cells expressing wild-type or mutant TRKB were stimulated with 1 ng/ml recombinant BDNF and analyzed for phospho- (p) ERK and total ERK. For all panels, the numbers underneath indicate quantification of the phospho-specific signals, normalized to the total signals and relative to those of wild-type TRKB.

Mentions: We have previously shown that TRKB-mediated oncogenic transformation of RIE-1 cells critically depends on TRKB kinase activity [25], [26]. In search of a biochemical explanation for the unanticipated results described above, we determined whether the cancer-derived TRKB mutants differ from wild-type TRKB in their responsiveness to BDNF. To measure TRKB activation, we used RIE-1 cells expressing wild-type or mutant TRKB but no ligand, and stimulated the cells with a physiologically relevant range of recombinant BDNF. In line with our previous studies [25], [26], this induced autophosphorylation of wild-type TRKB (Figure 4A and 4B), and led to the activation of two major downstream signaling pathways [22]: the PI3K pathway (resulting in phosphorylation of AKT/PKB; Figure 4C) and the MAPK pathway (resulting in phosphorylation of MAPK/ERK; Figure 4D). Exposure to 1 ng/ml BDNF was sufficient to elicit wild-type TRKB autophosphorylation and activate the MAPK pathway, whereas higher concentrations of BDNF were required to activate AKT (Figure 4B,C,D and data not shown). TRKBL138F and TRKBP507L responded to BDNF similarly to wild-type TRKB. Consistent with their inability to suppress anoikis, TRKBT695I was only partially activated by BDNF, while TRKBD751N was completely unresponsive to BDNF, identical to kinase-inactive TRKBK588M (Figure 4). These results show that TRKBT695I and TRKBD751N display reduced responsiveness to BDNF stimulation in rat epithelial cells, whereas TRKBL138F and TRKBP507L behave indistinguishably from wild-type TRKB.


Functional characterization of human cancer-derived TRKB mutations.

Geiger TR, Song JY, Rosado A, Peeper DS - PLoS ONE (2011)

Responsiveness of human cancer-derived TRKB mutants to BDNF.(A) RIE-1 cells expressing wild-type or mutant TRKB were stimulated with 10 ng/ml recombinant BDNF and analyzed for phospho-tyrosine (pY) and total TRK content by immunoprecipitation (IP) and subsequent immunoblot (IB) analysis. (B) RIE-1 cells expressing wild-type or mutant TRKB were stimulated with 1 ng/ml recombinant BDNF and analyzed for phospho (p) and total TRK. (C) RIE-1 cells expressing wild-type or mutant TRKB were stimulated with 10 ng/ml recombinant BDNF and analyzed for phospho- (p) AKT and total AKT. PI3K inhibitor LY294002 was applied to confirm the identity of the pAKT signal indicated by the arrowhead. Asterisk (*) indicates a non-specific band. The samples loaded in lanes two and three serve as controls and were derived from a replicate experiment performed under identical conditions. (D) RIE-1 cells expressing wild-type or mutant TRKB were stimulated with 1 ng/ml recombinant BDNF and analyzed for phospho- (p) ERK and total ERK. For all panels, the numbers underneath indicate quantification of the phospho-specific signals, normalized to the total signals and relative to those of wild-type TRKB.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3040757&req=5

pone-0016871-g004: Responsiveness of human cancer-derived TRKB mutants to BDNF.(A) RIE-1 cells expressing wild-type or mutant TRKB were stimulated with 10 ng/ml recombinant BDNF and analyzed for phospho-tyrosine (pY) and total TRK content by immunoprecipitation (IP) and subsequent immunoblot (IB) analysis. (B) RIE-1 cells expressing wild-type or mutant TRKB were stimulated with 1 ng/ml recombinant BDNF and analyzed for phospho (p) and total TRK. (C) RIE-1 cells expressing wild-type or mutant TRKB were stimulated with 10 ng/ml recombinant BDNF and analyzed for phospho- (p) AKT and total AKT. PI3K inhibitor LY294002 was applied to confirm the identity of the pAKT signal indicated by the arrowhead. Asterisk (*) indicates a non-specific band. The samples loaded in lanes two and three serve as controls and were derived from a replicate experiment performed under identical conditions. (D) RIE-1 cells expressing wild-type or mutant TRKB were stimulated with 1 ng/ml recombinant BDNF and analyzed for phospho- (p) ERK and total ERK. For all panels, the numbers underneath indicate quantification of the phospho-specific signals, normalized to the total signals and relative to those of wild-type TRKB.
Mentions: We have previously shown that TRKB-mediated oncogenic transformation of RIE-1 cells critically depends on TRKB kinase activity [25], [26]. In search of a biochemical explanation for the unanticipated results described above, we determined whether the cancer-derived TRKB mutants differ from wild-type TRKB in their responsiveness to BDNF. To measure TRKB activation, we used RIE-1 cells expressing wild-type or mutant TRKB but no ligand, and stimulated the cells with a physiologically relevant range of recombinant BDNF. In line with our previous studies [25], [26], this induced autophosphorylation of wild-type TRKB (Figure 4A and 4B), and led to the activation of two major downstream signaling pathways [22]: the PI3K pathway (resulting in phosphorylation of AKT/PKB; Figure 4C) and the MAPK pathway (resulting in phosphorylation of MAPK/ERK; Figure 4D). Exposure to 1 ng/ml BDNF was sufficient to elicit wild-type TRKB autophosphorylation and activate the MAPK pathway, whereas higher concentrations of BDNF were required to activate AKT (Figure 4B,C,D and data not shown). TRKBL138F and TRKBP507L responded to BDNF similarly to wild-type TRKB. Consistent with their inability to suppress anoikis, TRKBT695I was only partially activated by BDNF, while TRKBD751N was completely unresponsive to BDNF, identical to kinase-inactive TRKBK588M (Figure 4). These results show that TRKBT695I and TRKBD751N display reduced responsiveness to BDNF stimulation in rat epithelial cells, whereas TRKBL138F and TRKBP507L behave indistinguishably from wild-type TRKB.

Bottom Line: Unexpectedly, both colon cancer-derived mutants, TRKB(T695I) and TRKB(D751N), displayed reduced activity compared to that of wild-type TRKB.Consistently, upon stimulation with the TRKB ligand BDNF, these mutants were impaired in activating TRKB and its downstream effectors AKT and ERK.In conclusion, we fail to detect any gain-of-function of four cancer-derived TRKB point mutations.

View Article: PubMed Central - PubMed

Affiliation: Division of Molecular Genetics, Netherlands Cancer Institute, Amsterdam, the Netherlands.

ABSTRACT
Cancer originates from cells that have acquired mutations in genes critical for controlling cell proliferation, survival and differentiation. Often, tumors continue to depend on these so-called driver mutations, providing the rationale for targeted anticancer therapies. To date, large-scale sequencing analyses have revealed hundreds of mutations in human tumors. However, without their functional validation it remains unclear which mutations correspond to driver, or rather bystander, mutations and, therefore, whether the mutated gene represents a target for therapeutic intervention. In human colorectal tumors, the neurotrophic receptor TRKB has been found mutated on two different sites in its kinase domain (TRKB(T695I) and TRKB(D751N)). Another site, in the extracellular part of TRKB, is mutated in a human lung adenocarcinoma cell line (TRKB(L138F)). Lastly, our own analysis has identified one additional TRKB point mutation proximal to the kinase domain (TRKB(P507L)) in a human melanoma cell line. The functional consequences of all these point mutations, however, have so far remained elusive. Previously, we have shown that TRKB is a potent suppressor of anoikis and that TRKB-expressing cells form highly invasive and metastatic tumors in nude mice. To assess the functional consequences of these four TRKB mutations, we determined their potential to suppress anoikis and to form tumors in nude mice. Unexpectedly, both colon cancer-derived mutants, TRKB(T695I) and TRKB(D751N), displayed reduced activity compared to that of wild-type TRKB. Consistently, upon stimulation with the TRKB ligand BDNF, these mutants were impaired in activating TRKB and its downstream effectors AKT and ERK. The two mutants derived from human tumor cell lines (TRKB(L138F) and TRKB(P507L)) were functionally indistinguishable from wild-type TRKB in both in-vitro and in-vivo assays. In conclusion, we fail to detect any gain-of-function of four cancer-derived TRKB point mutations.

Show MeSH
Related in: MedlinePlus